Biosynthesis of multiple forms of β-endorphin in the reptile intermediate pituitary |
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Authors: | Robert M Dores Annmarie Surprenant |
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Institution: | University of Colorado, School of Medicine, Department of Physiology, Denver, CO 80262, USA |
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Abstract: | Fractionation of the β-endorphin-sized material from freshly dissected reptile intermediate pituitaries by ion exchange chromatography on sulfopropyl Sephadex (SP) revealed at least three distinct forms of immunoreactive β-endorphin. These forms eluted at 0.25 M NaCl, 0.28 M NaCl, and 0.32 M NaCl and represent respectively, 6%, 65% and 29% of the total immunoreactivity. Only the 0.28 M NaCl peak and the 0.32 M NaCl peak exhibited naloxone reversible opiate bioactivity when tested in the isolated guinea pig ileum bioassay system; taking into account the molar amount of immunoreactive peptides the 0.32 M NaCl peak was 6 fold more potent than the 0.28 M NaCl peak. Intermediate pituitaries in culture were incubated with either 3H]tyrosine, 3H]arginine, or 35S]methionine for periods up to 24 hours and β-endorphin-sized peptides were prepared by immunoprecipitation and gel filtration. Fractionation of the labeled β-endorphin-sized peptides by ion exchange chromatography yielded profiles nearly identical to the immunoassay analyses of freshly dissected tissue. Further analysis of the major labeled forms of reptile β-endorphin by chromatography on Sephadex G-50 equilibrated in 6 M guanidine HCl indicated that the 0.32 M NaCl peak had an apparent molecular weight of 3500±100 and the 0.28 M NaCl peak had an apparent molecular weight of 3200±100. Furthermore, pulse/chase experiments showed that the 0.32 M NaCl peak was the precursor for the 0.28 M NaCl peak. These results coupled with the relative opiate bioactivities of the major forms argue that the principal post-translational modification of reptile β-endorphin is COOH-terminal proteolytic cleavage. |
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Keywords: | β-Endorphin Opiate activity Biosynthesis Reptile intermediate pituitary |
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